Article Figures & Data
Figures
- FIG. 1.
Chromosomal contexts of the 10-kb regions containing genes encoding three putative ABC transporters which may participate in iron acquisition in GAS. Red arrow, lipoprotein gene; blue arrow, ATP-binding protein gene; green arrow, permease gene; pink arrow, hypothetical protein of unknown function black arrow, protein with known or inferred function. Above each arrow is the spy number assigned to the corresponding ORF in the M1 genome sequence (10). (A) ORFs around the htsABC transporter. Known or inferred functions are as follows: shp, heme-binding protein; 1791 and 1790, transporter proteins with transmembrane and ATP-binding domains. (B) ORFs around the spy0386-to-spy0383 transporter. Inferred functions are as follows: 0388, UDP-MurNac-tripeptide synthetase; 0380, manganese-dependent inorganic pyrophosphatase; 0379, pyruvate-formate lyase activating enzyme. (C) ORFs around the mtsABC transporter. Inferred functions are as follows: 0447, 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase; 0450, metal-dependent transcriptional regulator; 0457, peptidyl-prolyl cis-trans-isomerase.
- FIG. 2.
Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of purified recombinant HtsA, Spy0385, and MtsA. The gel was stained with Coomassie brilliant blue.
- FIG. 3.
HtsA binds heme. (A) UV-visible absorption spectrum of 50 μM HtsA in THCl. (B) Reductive spectra of pyridine hemochrome derived from HtsA (solid curve) and purified recombinant M. tuberculosis KatG (open circles) obtained as described in Materials and Methods. The spectra were normalized for comparison.
- FIG. 4.
Reconstitution of holo-HtsA. Purified HtsA, 82% of which was in apo-HtsA form, was incubated with heme, and the protein was separated from free heme by gel filtration using a Sephadex G-25 column. Presented are the optical absorption spectra of the untreated (dashed curve) and treated (solid curve) HtsA samples, both at 10 μM. The absorption peak at 408 nm is the Soret absorption peak of the bound heme.
- FIG.5.
Levels of iron, manganese, and zinc in purified Spy0385, MtsA, and HtsA proteins as measured by ICP-MS. Shown are concentrations of iron (A), manganese (B), and zinc (C) in buffer (THCl), Spy0385, MtsA, HtsA, and the negative-control protein Spy1294 (putative maltose/maltodextrin binding protein), each at 100 μM in the buffer.
- FIG.6.
(A) Effects of growth conditions on transcription of the gyrA, htsA, mtsA, and spy0385 genes. TaqMan assays were carried out with RNA isolated from GAS grown to an OD600 of 0.2 in metal cation-replete THY (open bars), Fe3+-depleted and Ca2+-, Zn2+-, Mn2+-, and Mg2+-replete DTHYM (solid bars), or Fe3+-, Ca2+-, Zn2+-, and Mn2+-depleted DTHYMg (hatched bars). The mean copy number of each indicated gene transcript in 30 ng of total RNA is shown. Error bars, standard deviations. (B) TaqMan analysis of htsA, mtsA, and spy0385 transcription in GAS grown in THY (open bars) or heparinized human blood (solid bars). GAS bacteria were harvested from THY at an OD600 of 0.4 or from human blood after a 4-h incubation. Levels of gene transcripts were normalized to the level of the transcript derived from the gyrA gene. Data are means ± standard deviations of triplicate measurements.
- FIG. 7.
Assessment of cell surface location of HtsA (top), Spy0385 (center), and MtsA (bottom) by flow cytometry. MGAS5005 (serotype M1) cells harvested at an OD600 of 0.4 were treated with a specific rabbit polyclonal antibody (red histograms) or a control rabbit anti-Sla antibody (histograms with solid black lines), stained with a phycoerythrin-conjugated donkey anti-rabbit IgG secondary antibody (histograms with dotted lines), and analyzed by flow cytometry.
Tables
- TABLE 1.
Primers, vectors, cloning sites, and recombinant plasmids in gene cloning
ORF Primers Vector/cloning sites Plasmid His tag spy0385 5′-ACCATGGGTGGTAATCAAGCAACTAATC-3′ pET-His2/NcoI, EcoRI pLP385 + 5′-CGAATTCTTAGTTTTCACTTGATAAGATTG-3′ mtsA 5′-ACCATGGGTTCGTCAACTGGCACTAAAAC-3′ pET21d/NcoI, EcoRI pLP453 − 5′-CGAATTCTTATTTTGCTAGACCTTCAG-3′ htsA 5′-ACCATGGGTGGTAATCAAGCAACTAATC-3′ pET-His2/NcoI, EcoRI pLP1795 + 5′-CGAATTCTTAGTTTTCACTTGATAAGATTG-3′ - TABLE 2.
Oligonucleotide primers and fluorescent probes used to quantitate cDNA
Gene Forward primer (5′-3′) Reverse primer (5′-3′) Probea (5′-3′) htsA CAGCACCCTAAAACGGCTAAA GGTCACAGATATCAACCACAGCAA AGACTGAACAGCAGAGAATTGTAGCCACTTCG mtsA GTCGTGGCAACCAATTCAATTATT ACAATGCTGTGCAGATCGATTT CCGACATGACAAAAGCTATTGCTGGTGA Spy0385 CGCAAACGTGATTATTTACAAGTGTT TTTCCAATCTTTTAACCACTTCTTGG TCTGACTTCGGCCGCATCTTTAACAAAGA gyrA CGACTTGTCTGAACGCCAAA TTATCACGTTCCAAACCAGTCAA CGACGCAAACGCATATCCAAAATAGCTTG ↵ a Covalently linked at the 5′ end to 5-carboxyfluorescein and at the 3′ end to N, N, N-tetramethyl-6-carboxyrhodamine.
